Tumor necrosis factor-a (TNF1) has potent, direct antiviral activity but has not been exploited as an antiviral therapy because of its systemic toxic effects. We have observed that primary human liver cells and hepatoma cells treated with low levels of TNF1 are resistant to infection with the hepatitis C virus (HCV) in vitro. This effect is dependent on TNF1 interaction with its receptor and is seen in the absence of detectable IFN1 or IFN2 gene expression. Sindbis virus, an alphavirus, infects Huh-7 hepatoma cells and is insensitive to TNF1 pretreatment. However, TNF1 synergizes with IFN2 to block Sindbis virus infection. We propose to use state- of-the-art techniques to identify key mediators of TNF1's antiviral effects alone and in combination with IFN2. We propose three Specific Aims. In the first Aim, we will test the requirements for signal transduction through the major pathways activated by TNF1 binding to its receptor, using pharmacologic inhibitors as well as dominant negative regulators of TNF1 signal transduction. In the second Aim, we will use novel virological and cell biological tools to define the stages in HCV infection that are inhibited following TNF1 treatment. In the third Aim, we will use microarray and next-generation sequencing approaches to define the effects of TNF1 on the transcriptome, identify candidate genes that may mediate the development of an antiviral state in TNF1- treated cells, and test the importance of these genes by knockdown and overexpression studies. The long-term goals of this research are to identify specific cellular pathways that mediate TNF1's antiviral activity, and to determine whether it is possible to isolate these activities from TNF1's systemic toxic effects.